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WO2004016785A1 - Methode d'examen de la dermatite atopique - Google Patents

Methode d'examen de la dermatite atopique Download PDF

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Publication number
WO2004016785A1
WO2004016785A1 PCT/JP2003/009999 JP0309999W WO2004016785A1 WO 2004016785 A1 WO2004016785 A1 WO 2004016785A1 JP 0309999 W JP0309999 W JP 0309999W WO 2004016785 A1 WO2004016785 A1 WO 2004016785A1
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WIPO (PCT)
Prior art keywords
gene
seq
genes
indicator
group
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PCT/JP2003/009999
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English (en)
Japanese (ja)
Inventor
Mikito Itoh
Kaoru Ogawa
Akira Shinagawa
Hajime Sudo
Hideoki Ogawa
Chisei Ra
Kouichi Mitsuishi
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Genox Research Inc
Juntendo University
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Genox Research Inc
Juntendo University
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Priority to JP2004528848A priority Critical patent/JPWO2004016785A1/ja
Priority to AU2003252418A priority patent/AU2003252418A1/en
Publication of WO2004016785A1 publication Critical patent/WO2004016785A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for detecting atopic dermatitis.
  • Allergic diseases such as atopic dermatitis are considered to be multifactorial diseases. These diseases are caused by the interaction of the expression of many different genes, and the expression of these individual genes is affected by multiple environmental factors. Therefore, it is very difficult to elucidate a specific gene that causes a specific disease.
  • allergic diseases are thought to be related to the expression of mutated or defective genes, or overexpression or decreased expression of specific genes. Understanding the role of gene expression in disease requires an understanding of how genes are involved in pathogenesis and how external stimuli, such as drugs, alter gene expression. is there.
  • allergen-specific IgE measurement, leukocyte histamine release test, and lymphocyte blastogenesis test.
  • the presence of allergen-specific IgE is evidence of an allergic reaction to the allergen.
  • some patients may not always be able to detect allergen-specific IgE.
  • diagnosis Testing must be performed for all of the arenolegens required for The leukocyte histamine release test and the lymphocyte blastogenesis test are methods for observing the response of the immune system to allergens using iiro. These methods are complicated in operation.
  • a method for utilizing the immune response observed when a patient is actually contacted with an allergen to diagnose allergens is also known. Prick tests, scratch 'tests, patch' tests, intradermal reactions, or provocation tests are included in this type of test. While these tests can directly diagnose a patient's allergic reaction, they can be described as tests involving invasive exposure of the subject to an allergen.
  • test methods have been attempted to prove the involvement of allergic reactions regardless of the allergen.
  • a high serum IgE level may indicate that the patient has an allergic reaction.
  • the serum IgE value is information corresponding to the total amount of allergen-specific IgE. It is easy to determine the total amount of IgE regardless of the type of allergen, but patients with diseases such as non-atopic bronchitis asthma may have low IgE levels.
  • the present invention has been made in view of such a situation, and an object of the present invention is to provide a new index that enables a test for atopic dermatitis. Further, the present invention provides a method for testing atopic dermatitis based on the index, and a therapeutic drug for atopic dermatitis. An object of the present invention is to provide a method for screening a complement compound.
  • the present inventors have intensively studied to solve the above problems.
  • we clarified genes that showed a difference in the expression level between the rash in the same patient as the rash in atopic dermatitis patients and the rash in the patients with atopic dermatitis and the skin of healthy subjects.
  • we elucidating the relationship with the atopic dermatitis reaction we thought that a new target for the treatment of atopic dermatitis could be found.
  • the present inventors have found that the eruption in the same patient as the eruption of atopic dermatitis patients and the rash between the eruption of atopic dermatitis patients and the skin of healthy subjects We searched for genes with different expression status.
  • the skin tissue of the subject was selected as a biological sample for comparing gene expression states.
  • Many infiltration of lymphocytes, etc., which are important for the pathogenesis is infiltrated into skin tissue specimens where inflammation has actually occurred, and analysis of gene expression in local skin will lead to elucidation of the pathology of atopic dermatitis. Conceivable.
  • the present inventors compared the expression profiles of the atopic part of the same atopic dermatitis patient's rash area and the gene expressed in the skin of healthy subjects using a gene chip. Genes with expression fluctuations of two times or more were selected, and the expression levels of the genes were measured. Then, it was confirmed that the expression of an indicator gene selected from the group described in any of the following a) to fluctuated. Furthermore, the present inventors analyzed the expression level of the mouse counterparts of these indicator genes in a mouse dermatitis model, and identified any one of A) to D) as an indicator gene related to mouse dermatitis. The indicator genes described in the group were selected.
  • a part of the nucleotide sequence of each indicator gene described in any of a) to is known as EST.
  • a part of the nucleotide sequence of each indicator gene described in any of A) to D) is known as EST.
  • the functions of the proteins encoded by the indicator genes a) to d) are described in the papers described in the Reference section of Data 1, Data 2, Data 5, and Data 6 described below.
  • the functions of the proteins encoded by each of the indicator genes A) to .D) are as follows: Data 3, Data 4, Data 11 and Data 12. It is described in the paper shown in the rence section.
  • the present inventors have made it possible to diagnose atopic dermatitis or psoriasis by using the expression level of each indicator gene or the activity of the protein encoded by each indicator gene as an indicator.
  • the present inventors have found that screening of therapeutic agents for the disease is possible, and completed the present invention. That is, the present invention relates to the following methods for detecting atopic dermatitis and a method for screening candidate compounds for a therapeutic agent for atopic dermatitis.
  • a method for detecting atopic dermatitis comprising the following steps (1) to (3), wherein the indicator gene is selected from the group described in any of the following a) to The method that is the gene.
  • step (1) The expression level measured in step (1) was collected from the rash-free area of the same subject as a control when the indicator gene was the gene described in a) or b) Comparing the expression level of the indicator gene in the biological sample with the expression level of the indicator gene in the raw material of a healthy subject as a control, if the indicator gene is a gene described in c) or d) , and
  • step (2) when the indicator gene is a gene described in a) or c), the expression level is higher than that in the control, and when the indicator gene is b) or d) A) determining that the subject has atopic dermatitis when the expression level of the gene described in
  • AI458014 (NM_018509 / SEQ ID NO: 9), ⁇ 127950-020183 / SEQ ID NO: 10), AI801545 (NM— 021928 / SEQ ID NO: 11), AI823872 (NM-022136 / SEQ ID NO: 1)
  • AI570209 (NM-0124329 / SEQ ID NO: 13), AA417813 (NM-024636 / SEQ ID NO: 14), T62854 (NM-024829 / SEQ ID NO: 15), AI129310 (NM-025113 / SEQ ID NO: 16) ), AA601529 (NM—030938 / SEQ ID NO: 17), AI913548 (NM-1032022 / SEQ ID NO: 18), AI818662 (NM—032488 / SEQ ID NO: 19), AA532392 (NlVi__032731 / SEQ ID NO: 20), AA633203 (NM-033255 / SEQ ID NO: 21), AI859144 (AB032953 / SEQ ID NO: 22), A1819863 (SEQ ID NO: 23), W24320 (SEQ ID NO: 24), AI859620 (AF293462 / SEQ ID NO: 25), AA191741 (AK021498 / SEQ ID NO:
  • AI935239 (SEQ ID NO: 40), AI718763 (SEQ ID NO: 41), ⁇ 60613 (SEQ ID NO: 42), ⁇ 424160 (SEQ ID NO: 43), AW005250 (SEQ ID NO: 44), AI43I800 (SEQ ID NO: 45), AI088609 (SEQ ID NO: 46), AI971000 (SEQ ID NO: 47), All 3 9470 (SEQ ID NO: 48), AA772360 (SEQ ID NO: 49), AI805006 (SEQ ID NO: 50), AW014155 (SEQ ID NO: 51), W68630 (SEQ ID NO: 52), AI369347 (SEQ ID NO: 53), AA160156 (SEQ ID NO: 54), AA422178 (SEQ ID NO: 55), AI671741 (SEQ ID NO: 56) A gene containing any of the base sequences of SEQ ID NO: 57, AI674565 (SEQ ID NO: 57), H87671 (SEQ ID NO: 58),
  • a group of indicator genes consisting of the following genes, whose expression level in the rash area is lower than that in the non-rash area in patients with atopic dermatitis: GenBank accession numbers AI738919 (NM-0332622), AL047586 (NM — 005105), All 89838 (NM—021571), AI131052 (NM_017786), AA628405 (NM-0133254), AI094860 (NM—001719), AA621478 (NM_004673) AI889132 (NM_021101), AI377221 (NM_006434), AA14213 (NM1) 014476), AI347165 (NM-002441), AI741530 (NM-013261), AI343258 (NM-014439), H61590 (NM-004711), AI149693 (NM-005678), AA031286 (NM-003749), AW007116 (NM — 003740), A I632567 (NM— 014553
  • An index gene group consisting of the following genes, whose expression level in the eruption area of atopic dermatitis patients is higher than that in healthy subjects: the following GenBank accession numbers: AA196189, AI588981, H17272, R11505, AA286909 , AI655892, W60377.
  • the index genes of a) are the following GenBank accession numbers: AI680350, AI261490, N22028, AI458014, AI760613, AA191741, AI129310, W44526, AW014646, AI913548, AI768116, AA652869, AW005250, AI655668, AA765843 , A 1632223 ⁇ AI807277, AI763378, AA196201, AI301935, AI431800, AI968085, AI47211 AI088609, AI983204, AW014155, AI718763, AA424160, N32858, AI971000, AI655719, AI817147, AI986192, AI139470, AI935239, AA948682, AA028
  • the method according to [1] which is any gene selected from genes containing any of the nucleotide sequences represented by H06350, AI67174K, AI983994, and AI039915.
  • the indicator genes of a) are the following GenBank accession numbers W24320, AI7615 20, AA142976, N45367, AI076809, AA532392, AI472143, AA744772, AI67 4565, AI805006, AI570209, AA160156, AA669106, AI857997.
  • [4] indicator genes of b) has the following GenBank of ⁇ click session number R53594, AW006 208, N37065, AI934361 , AI524912, All 50703 s AA621478. AI743780, AI560 147, AI057637, AI347165, AW006912. AA583350, AI985094, All 89838, A A161496, AI814253, W25633, AA031286, AA724373, AA541564, AI709055. , AI741934, and AI832193, the method according to [1], which is any gene selected from genes containing any of the nucleotide sequences represented by AI831193 and AI832193.
  • the indicator genes of b) are the following GenBank accession numbers AA458648, W01370, W85913, AI377221, AA632130, AI267333, AL119027, AI741530, AI34 6282, AA662105, AI969486, AA886888, AW02105U AA142913, AA723692, AI703114, All 86548, AA284268 S H2350S, All 31052, W60377, AA947123, AI819048, AI979261, W56090, AI989530, AI806754, AI738919, AI598222, W35214, AL047586, AI650542, AI149693, AW007116, AI676241, AI676059, AI670708, AI870733 AI492388, and any gene selected from genes containing any of the nucleotide sequences represented by W68180 [1] The described method.
  • the index genes of c) are the following GenBank accession numbers: AI68863U AI351607, AI828042, AL046653, AI432451, All 40989, AI689755, AA877124, AA428312, AI375097, AI393240, N94985, AA468768, AA704465, AI332430s AI681436, AI690823, AI741934, R07848, AA669135, AA88387A, AA6298764, AA6298764A AI791189, AW00 7121s AA705219, AA810719, AA767372, AI744663, AI225084, AA602620, AI680822, and any one of the genes selected from genes containing any of the nucleotide sequences represented by ⁇ 733467.
  • the index genes of c) are the following GenBank accession numbers: AI588981, H17272, R11505, W60377, AI636016, AI888493, AI031771, AI076830, AW023597, AA026238, M79158, AI937383, AA603217, AA766886, and AI344053 [1]
  • GenBank accession numbers AI588981, H17272, R11505, W60377, AI636016, AI888493, AI031771, AI076830, AW023597, AA026238, M79158, AI937383, AA603217, AA766886, and AI344053 [1]
  • the method according to [1] which is any gene selected from genes containing any of the nucleotide sequences shown in [1].
  • the indicator genes of d) are the following GenBank accession numbers: AI821404, AA424943, F28162, AI650353, AI937421, AL039926, AW016780, AI796988, W72331, AA429326, AA651724, AI743715, AI377043, AI670876, N32483 , R50 231, AI300085, W86423, AI924323, AI307802, AA65906 AI570212, AA52 3434, AI094787, AI050752, AI269126, AI651732, AI283548, AI763004, AA 702419 ⁇ AI540161, AI832243, AL043717, AI954900, AI986085, AI165654 , AI972953, AL041424, AI963104, AI986246, H79244, AI751438, AA610659, AA436185, W26884, AI800110, AW00522K AI754719,
  • the index genes of d) are the following GenBank accession numbers: AI680350, AA01 9641, AA019557, AI816806, AA534163, H16294, AA004689, AI671885, AI 244908, AA053401, W72665, H60397, AA525157, AI888485, H87064 , R07844, T57077, AA508138, AI800470, AI458464, AI431778, AA632649, AA523939, and any one of the genes selected from genes containing any of the nucleotide sequences shown in AI681868 (1).
  • test reagent for atopic dermatitis comprising a polynucleotide containing the base sequence of the indicator gene or an oligonucleotide having a base sequence complementary to its complementary strand and having a length of at least 15 bases
  • indicator gene is any gene selected from the group according to any of a) to [1].
  • a reagent for atopic dermatitis testing comprising an antibody recognizing a protein encoded by an indicator gene, wherein the indicator gene is selected from the group described in any of a) to a) in [1]. Any of the following genes for atopic dermatitis testing
  • a method for screening a therapeutic drug for atopic dermatitis comprising the following steps, wherein the indicator gene is selected from the group described in any of a) to a) in [1].
  • a screening method that is the gene.
  • a marker gene for the a) group or c) group is a compound that reduces the expression level of the gene, and b) group or d) selecting a compound that increases the expression level of the marker gene of the group
  • an atopy comprising a polynucleotide comprising the nucleotide sequence of the indicator gene, or an oligonucleotide having a nucleotide sequence complementary to the complementary strand thereof and having a length of at least 15 nucleotides; and a cell expressing the indicator gene.
  • kits which is any gene selected from the group described in any of the above-mentioned.
  • a kit for screening a candidate drug for treating atopic dermatitis comprising an antibody recognizing a protein encoded by an indicator gene, and a cell expressing the indicator gene, the kit comprising an indicator gene Is a gene selected from the group according to any of a) to [1] in [1].
  • An atopic dermatitis model animal comprising a transgenic non-human vertebrate with increased expression intensity in the skin of an indicator gene or a gene functionally equivalent to the indicator gene, wherein the indicator gene is as defined in [1].
  • An atopic dermatitis model animal consisting of a transgenic non-human vertebrate with reduced expression intensity in the skin of an indicator gene or a gene functionally equivalent to the indicator gene, wherein the indicator gene is (1) A model animal that is any gene selected from the group described in b) or d) above, and also in the following B) or D).
  • An indicator gene group consisting of the following genes, whose expression level in the auricle skin of mite-allergen-sensitized mice is lower than that of the mite-allergen-unsensitized mice: cla udin 1 Curated Ortholog (AF072127), eukaryotic elongation factor-2 kinase (U9384 8), UI-M-BH0-aiy-b-12-0-UI.sl (AI852760 / SEQ ID NO: 16 2), RIKEN cDNA 24 00004E04 gene (AI846720 / SEQ ID NO: 16) 3), ub62bl0.xl Mus musculus cDNA (AI462309 / SEQ ID NO: 164), UI-M-BHl-alo-a-04-0-UI.sl (AW047645 / SEQ ID NO: 165), and Mus musculus cDNA (238618 / SEQ ID NO: 1 6 6)
  • a method for producing an atopic dermatitis model animal comprising a step of administering the component according to any one of the following 1) to 4) to a mouse.
  • An inducer for inducing atopic dermatitis in mice comprising as an active ingredient the ingredient according to any of 1) to 4) in [22].
  • a method for screening a therapeutic agent for atopic dermatitis comprising the following steps, wherein the indicator gene is any one of a) to c) in [1], and A) and C in [18 ']. ), And a screening method which is any gene selected from the group described in B) and D) in [20], or a gene functionally equivalent to the indicator gene.
  • the indicator genes of the groups a), c), A) and C) are compounds which reduce the expression level of the genes, and b) , D), B), and D) for the indicator gene, Step of selecting a compound that increases the expression level
  • a method for screening a therapeutic agent for atopic dermatitis comprising the following steps, wherein the indicator gene is any one selected from the group described in any of a) to a) in [1]. Or a screen-jung method that is a gene functionally equivalent to the indicator gene.
  • a method for screening a therapeutic agent for atopic dermatitis comprising the following steps, wherein the indicator gene is selected from the group described in any of (a) to (d) in (1).
  • a screening method that is a gene or a gene that is functionally equivalent to an indicator gene.
  • atopic dermatitis comprising as an active ingredient a compound obtainable by the screening method according to any one of [14], [24], [25], and [26]; Remedy.
  • [28] Contains an indicator gene or a part of its antisense DNA as an active ingredient A therapeutic agent for atopic dermatitis, wherein the indicator gene is any of the genes selected from the group described in any of a) or c) in [1]. .
  • a therapeutic agent for atopic dermatitis comprising as an active ingredient an antibody recognizing a protein encoded by an indicator gene, wherein the indicator gene is any of a) or c) in [1].
  • a therapeutic agent for atopic dermatitis comprising as an active ingredient a marker gene or a protein encoded by the marker gene, wherein the marker gene is (b) or (d) in (1).
  • DNA chip which is at least one gene selected from the group consisting of:
  • the present invention provides a method for treating atopic dermatitis, comprising the step of administering a compound obtainable by the screening method according to any one of (14), (24), (25) and (26). Regarding treatment methods.
  • the present invention also provides a pharmaceutical composition for treating atopic dermatitis, comprising a compound obtainable by the screening method according to any one of [14], [24], [25] and [26]. Use in the manufacture of
  • the present invention relates to a method for treating atopic dermatitis, comprising a step of administering the following component (i) or (ii).
  • the present invention relates to the use of the following component (i) or (ii) in the manufacture of a pharmaceutical composition for treating atopic dermatitis.
  • the present invention relates to a method for treating atopic dermatitis, comprising a step of administering the following component (iii) or (iv).
  • the present invention relates to the use of the following component (iii) or (iv) in the manufacture of a pharmaceutical composition for treating atopic dermatitis.
  • the present invention also relates to the following psoriasis detection method and screening method for a psoriatic therapeutic drug candidate compound.
  • a psoriasis test method comprising the following steps (1) to (3), wherein the indicator gene is any one selected from the group described in any of the following: Method.
  • step (1) The expression level measured in step (1) was collected from the rash-free area of the same subject as a control when the indicator gene was the gene described in i) or ii).
  • step (2) The expression level measured in step (1) was collected from the rash-free area of the same subject as a control when the indicator gene was the gene described in i) or ii).
  • step (3) A step of comparing the expression level of the indicator gene in the biological sample with the expression level of the indicator gene in a biological sample of a healthy subject as a control when the indicator gene is a gene described in iii) or i V) , and
  • An index gene group consisting of the following genes, whose expression level in the rash area is higher than that in the rash area in psoriatic patients: GenBank accession numbers W61185, R95872, AA007294 S AI348427 S W22165, AA577672, N42752 , AF034175, A A742438, AA398352, AI421812, AA195614, AI990409, AI34126K AI076192, AI985652, AA421326, AA280072, AA916868, AA234670 S AA056755, AI540 870, AA77910 AA011633, AA521489, AI394016, A394016, A394016, R40393, A40016, R40393 AI808807, AA134958, AA143794, AA151346, All 2 5673, AA948319, R97448, All 60811, AI452797, AI379080, AK02
  • An index gene group consisting of the following genes, whose expression level in the rash area of psoriasis patients is higher than that in healthy subjects: Accession numbers of the following GenBank: AA470061, AA446965, T5359K AA552006, ALl 19305, AI023320 , AI310139, AA029791, AI968310, H99215, N22028, AA48750K T90962, AA910404, AI 492412, AA993042, AA129756, AI950930, AI989841, AA069368, AI989772, AI989567, AI128216, AI688189, AI753316, H06246A, 686, A6 N52767, AI760332, N99568, AI70159K A A121732, T79942, AA203555, AA004443, AA126468, AI858054, AI384076, AI921873, T99531, AA005023
  • Genes comprising the following genes, whose expression levels in the rash area of psoriasis patients are lower than those in healthy subjects: the following GenBank accession numbers W3 3155, AA404418, AA284279, AA531023, AI022632 , W16645, AA011633, A1935353, AI671062, AI921885, AI566793, AI279946, AI291048, AI291314, R05297, AI984197, AA928770, AI916305, AI203206, AI632214, AI074020, AI978869, AI475680, AI191110, T92947, W73694, N38 , AI337300, AI741253, W30810, AA826176.
  • the indicator genes of i) are the following GenBank accession numbers W61185, R958 72, AA007294, AI348427, W22165, AA577672 N N42752, AF034175, AA742 438, AA398352, AI421812, AA195614, AI990409, AI341261, AI076192, AI985652, AA421326, AA280072, AA916868.
  • index genes of i) are GenBank accession numbers W24320, AI76 1520, AA142976, N45367, AI076809, AA532392, AI472143, AA744772, AI 674565, AI805006, AI570209, AA160156, AA669106, AI857997, AA417813.
  • the indicator genes of ii) are the following GenBank accession numbers N21096, AA66 1990, AI079545, AA455877, W7448U AA533275, N63913, AA129756, AI697569, AI989841, AA587950, AI340029, AI128216, H61109, AL048159 , H16 791, AI968274, AA524036, N51315, W72920, AI911559, AI743671, AA679 863, AI765692, AI634580, AA147751, AA173572, AI304339, AI973108, AA 149594, AI806338, AL040063, AI379772, AA102575, AI659533 R 25483 ⁇ AI864898, AL046941, AI743925, C14904, N49899, AI8 19282, AI376944, AI860751, AI76124 H43374, R32893, AI972123,
  • the indicator genes of ii) are the following GenBank accession numbers: AA72369
  • Indicator gene cluster 7] iii) has the following GenBank of ⁇ click session number AA470061, A A446965, T53591, ⁇ 552006 , AL119305, AI023320, AI310139, AA02979 AI968310, H99215 N N22028, AA487501, T90962, AA910404, AI492412 S AA 993042 , AA129756, AI950930, AI989841, AA069368, AI989772, AI989567, AI128216, AI688189, AI753316, H06219, AI686411, AI24664K AA524743, W85913, AW024527, N52767, AI760332, N99568, AI701591, AA121732, T79942A, A79732, T79942A , AI384076, AI921873, T9953 AA005023, AA513397, AI821405, AA292265
  • the indicator genes of iii) are the following GenBank accession numbers: AI588981, HI7272, R11505, W60377, AI636016, AI888493, AI031771, AI076830, AW023 597, AA026238, M79158, AI937383, AA603217, AA766886, The method according to [32], which is any gene selected from genes containing any of the nucleotide sequences represented by AI344053.
  • the indicator genes of iv) are the following GenBank accession numbers W33155, AA4 04418, AA284279, AA531023, AI022632, W16645, AA011633, AI935353, A1671062, AI921885, AI566793, AI279946, AI291048, AI291314, R05297, AI 984197, AA928770, AI916305, AI203206, AI632214, AI074020, AI978869, AI475680, AI191110, T92947, W73694, N38970, F04368, AA649208, AI337 300, AI741253, W30810, AA826176, H03969, AA058522, AI2006 011, AI760495, AA779265, AI217339, AI760366, AA417099, AI95045K or the gene according to [32], which is any gene selected from genes containing any of the nucleotide sequences represented by AI392S46
  • index genes of iv) are the following GenBank accession numbers: AI6S0350, A A019641, AA019557, AI816806, AA534163, H16294, AA004689, AI671885 AI244908, AA05340U W72665.H60397, AA525157, AI888485, H87064, R0
  • the method according to [32] which is any gene selected from genes containing any of the nucleotide sequences represented by 7844, T57077, AA508138, AI800470, AI458464, AI431778, AA632649, AA523939, and AI681868.
  • a psoriasis test reagent comprising a polynucleotide containing the nucleotide sequence of an indicator gene or an oligonucleotide having a nucleotide sequence complementary to a complementary strand thereof and having a length of at least 15 bases, wherein the indicator gene comprises: Is a gene selected from the group according to any one of the above [32] to [iv]. Iv).
  • a psoriasis detection reagent comprising an antibody recognizing a protein encoded by an indicator gene, wherein the indicator gene is selected from the group described in any of (32) to (iv)) A psoriasis test reagent that is any of the genes.
  • a psoriatic disease comprising a polynucleotide comprising the nucleotide sequence of the indicator gene, or an oligonucleotide having a nucleotide sequence complementary to the complementary strand thereof and having a length of at least 15 nucleotides, and a cell expressing the indicator gene;
  • kits for screening a psoriasis therapeutic candidate compound including an antibody recognizing a protein encoded by an indicator gene and cells expressing the indicator gene, wherein the indicator gene is [32] ]
  • a psoriatic model animal comprising a transgenic non-human vertebrate with an increased expression level in the skin of an indicator gene or a gene functionally equivalent to the indicator gene, wherein the indicator gene is the same as in [32].
  • a model animal which is any gene selected from the group described in group iii) group.
  • a psoriatic model animal comprising a transgenic non-human vertebrate with reduced expression intensity in the skin of an indicator gene or a gene functionally equivalent to the indicator gene, wherein the indicator gene is H in [32].
  • a method for screening a therapeutic agent for psoriasis comprising the following steps, wherein the indicator gene is any gene selected from the group described in any of (iv) to (iv) in (32), or A screening method in which the gene is functionally equivalent to the indicator gene.
  • a method for screening a therapeutic agent for psoriasis comprising the following steps, wherein the indicator gene is any one of the genes selected from the group according to any one of i) to iv) in [32]. Or a screening method that is a gene functionally equivalent to the indicator gene.
  • a therapeutic agent for psoriasis comprising as an active ingredient a compound obtainable by the screening method according to any one of [45], [53] and [54].
  • a therapeutic agent for psoriasis containing an indicator gene or a part of the antisense DNA as an active ingredient, wherein the indicator gene is selected from the group described in any of i) or iii) in [32] Therapeutic agents that are any of the genes that were performed.
  • a therapeutic agent for psoriasis comprising as an active ingredient an antibody recognizing a protein encoded by an indicator gene, wherein the indicator gene is selected from (i) or (iii) in (32).
  • An indicator gene or a protein encoded by the indicator gene is defined as an active ingredient.
  • a diagnostic DNA chip for psoriasis having a probe for measuring an indicator gene immobilized thereon, wherein at least one of the indicator genes described in [32] or selected from any of the groups iv) is selected.
  • DNA chip a kind of gene.
  • the present invention relates to a method for treating psoriasis, comprising a step of administering a compound obtainable by the screening method according to any one of [45], [53], and [54].
  • the present invention also relates to the use of a compound obtainable by the screening method according to any one of [45], [53] and [54] in the manufacture of a pharmaceutical composition for treating psoriasis.
  • the present invention relates to a method for treating psoriasis, comprising a step of administering the following component (1) or (2).
  • the present invention relates to the use of the following component (1) or (2) in the manufacture of a pharmaceutical composition for treating psoriasis.
  • the present invention relates to a method for treating psoriasis, comprising a step of administering the following component (3) or (4).
  • the present invention relates to the use of the following component (3) or (4) in the manufacture of a pharmaceutical composition for treating psoriasis.
  • an allergic disease is a general term for diseases associated with allergic reactions. More specifically, it can be defined as identifying an allergen, demonstrating a deep link between exposure to the allergen and the development of the lesion, and demonstrating an immunological mechanism for the lesion.
  • the immunological mechanism means that white blood cells show an immune response by stimulation of allergen. Allergen Examples thereof include mite antigens and pollen antigens.
  • Representative allergic diseases can include atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, or insect allergy.
  • Allergic diathesis is a genetic factor transmitted from a parent with an allergic disease to a child.
  • a familial allergic disease is also called an atopic disease, and the genetic factors that cause it are predisposed to atopy.
  • Atopic dermatitis is a generic term given to atopic diseases, especially those accompanied by skin inflammatory conditions.
  • AD indicator gene a gene that can be used as an indicator of atopic dermatitis.
  • a protein consisting of an amino acid sequence encoded by an AD indicator gene is called an AD indicator protein.
  • the AD indicator gene is used as a term indicating any one or more arbitrary genes selected from the genes described in a) to d) and A) to D).
  • genes selected from the following gene groups are preferable as the genes of the above-mentioned group a) or group b). These genes have higher expression levels in the eruption area of patients with atopic dermatitis than in the rash area of patients with atopic dermatitis.
  • group 1 or low b) Group 1 AD These genes are included in the indicator gene group and have no significant difference in their expression levels in the comparison between rash and rash in psoriasis patients.
  • These AD indicator genes are genes whose expression levels are specifically changed in patients with atopic dermatitis.
  • Preferred genes as group genes are: Accession numbers of the following GenBank: R53594, AW006208, N37065, AI934361, AI524912, AI150703, AA621478 AI743780, AI560 147, AI057637, AI347165, AW006912, AA583350, AI985094, AI189838, M161496, AI814253 , W25633, AA031286, AA724373, AA541564, AI709055, N91161, AW02558 4, H61590, AI094860, M628405, AA057445, AI799784, AA034418, AI972873, AA 629842, AI935541, H73606, AI334358, AI889132, AI889132, AI889132, AI889132, AI889132, AI8893 A gene containing any of the nucleotide sequences shown in
  • genes selected from the following gene groups are preferable as the genes of the above-mentioned group c) or group d). These genes are included in the AD index gene group, one of which is higher in the eruption area of patients with atopic dermatitis than in healthy subjects, c) one group, or one low d) group, and psoriasis. It is a gene for which no significant difference was found in the expression level in a comparison between a non-eruptive part of a patient and a healthy person. These AD indicator genes are also genes whose expression levels are specifically changed in patients with atopic dermatitis.
  • Genes preferred as group genes GenBank accession number AI8214 04, AA424943, F28162, AI650353, AI937421, AL039926, AW016780, AI796988, W 72331, AA429326, M651724, AI743715, AI377043, AI670876, N32483, R50231, AI300085 S W86423, AI924323, AI307802, AA659061, AI570212, AA523434 AI050752, AI269126, AI651732, AI283548, AI763004, M702419, AI540161, AI832243, AL043717, AI954900, AI986085, AI361654, AL119913, D63177, AI479 165, AI972953, AL041424, AI963104 S AI986246, H79244, AI751438,6 Any gene selected from genes containing any of the nucleotide sequences represented by AI800110, ATO05221,
  • a gene that can be used as an indicator of psoriasis is called a psoriasis indicator gene.
  • a protein consisting of an amino acid sequence encoded by a psoriasis indicator gene is called a psoriasis indicator protein.
  • the psoriasis indicator gene is used as a term indicating any one or more genes selected from the genes described in i) to iv).
  • the term is used as a term including both the AD indicator gene (and the AD indicator protein) and the psoriasis indicator gene (psoriasis indicator protein).
  • a gene selected from the following gene group is preferable as the gene of the group i) or the group ii).
  • These genes are included in the psoriatic index gene group (i) group-- or low ii)-the expression level in the rash area of psoriasis patients is higher than that in the non-rash area of psoriasis patients, and This gene did not show a significant difference in expression level between the rash and rash in inflamed patients.
  • These psoriasis indicator genes are genes whose expression levels are specifically changed in psoriatic patients.
  • Preferred genes as group genes are: Accession numbers W61185, R95872, M007294, AI348427, W22165, AA577672, N42752, AF034175, AA742438 AA398352, AI421812, AA195614.
  • genes selected from the following gene groups are preferable as the genes of the above group iii) or iv). These genes are included in the psoriatic index gene group in which the expression level in the eruption area of psoriasis patients is higher than that in healthy subjects iii) group 1 or lower iv). This is a gene for which no significant difference was found in the expression level between the rash and healthy subjects. These psoriasis indicator genes are also genes whose expression levels are specifically changed in psoriatic patients.
  • iii) Preferred genes as group genes are: Accession numbers of the following GenBank: M470061, AA446965, T53591, AA552006 S AL119305, AI023320, AI310139, AA029791 AI968310, H99215, N22028, M487501, T90962, M910404, AI492412, AA993042.
  • a part of the nucleotide sequence of the indicator gene in the present invention is known as EST.
  • the amino acid sequence encoded by the nucleotide sequence of the indicator gene in the present invention has been clarified.
  • GenBank registration numbers for obtaining partial nucleotide sequence data of the indicator gene are listed below together with the name of the indicator gene.
  • the full-length nucleotide sequence of the indicator gene can be obtained, for example, by in silico cloning.
  • the EST base sequence (query sequence) that constitutes a part of the indicator gene is checked against the vast amount of EST information accumulated in public databases. Based on the result of the collation, obtain other EST information whose base sequence matches the query sequence over a certain length. The obtained other EST information is used as a new query sequence, and the acquisition of other EST information is repeated. By repeating this operation, a set of multiple ESTs sharing a partial nucleotide sequence can be obtained. The set of ESTs is called a cluster.
  • the base sequence of the target gene can be clarified by superimposing the base sequences of ESTs constituting the cluster and integrating them into one base sequence.
  • those skilled in the art can design primers for PCR based on the nucleotide sequence determined by insiJ / cocloning. By confirming that a gene fragment with the designed length is amplified by RT-PCR using these primers, it is possible to confirm that a gene consisting of the determined nucleotide sequence actually exists. .
  • Northern blotting can be used to evaluate the results of in silico oral lingering. Perform Northern plotting using a probe designed based on the determined nucleotide sequence information. As a result, the above base sequence information and If a matching band can be detected, the presence of the gene having the determined base sequence can be confirmed.
  • a cDNA clone registered as an EST and provided with nucleotide sequence information is obtained, and the entire nucleotide sequence of the cDNA of the clone is determined. As a result, the full-length sequence of the cDNA may be revealed. At least, longer nucleotide sequences can be revealed. The length of the cDNA of the clone can be experimentally confirmed in advance if the structure of the vector is clear.
  • a method for obtaining a part whose base sequence is unknown based on the partial base sequence even when a clone to which the base sequence information of the EST is not available is known. For example, screening a cDNA library using EST as a probe may reveal a longer nucleotide sequence. If a library containing a large amount of full-length cDNA is used as a cDNA library, a full-length cDNA clone can be easily isolated. For example, a cDNA library synthesized based on the principle of the oligocap method is said to contain a large amount of full-length cDNA.
  • the RACE method is a typical technique for isolating a gene containing an unknown nucleotide sequence.
  • an oligonucleotide linker is artificially linked to the end of cDNA.
  • the nucleotide sequence of this oligonucleotide linker is known in advance. Therefore, a primer for PCR can be designed based on the region whose base sequence is already known as the EST and the base sequence information of the oligonucleotide linker. PCR using the primers designed in this way specifically synthesizes regions with unknown nucleotide sequences.
  • the method of testing for an allergic disease of the present invention measures the expression level of each indicator gene in a biological sample of a subject, and when the indicator gene is a gene described in a) or b) above, As a control, a biological sample taken from the rash of the same subject A step of comparing the expression level of the indicator gene with the expression level of the indicator gene in a biological sample of a healthy subject as a control when the indicator gene is a gene described in C) or d) above. Including. If the indicator gene is a gene described in a) or c) above, the subject is determined to be atopic dermatitis if the expression level is higher than that of the control. If the indicator gene is a gene described in b) or d) above, the subject is determined to be atopic dermatitis if the expression level is lower than that of the control.
  • the psoriasis test method of the present invention measures the expression level of each indicator gene in a biological sample of a subject, and when the indicator gene is a gene described in i) or ii) above, the same as a control Expression level of the indicator gene in a biological sample collected from the rash of the subject, and if the indicator gene is a gene described in iii) or iv) above, a biological sample of a healthy subject as a control And comparing with the expression level of the indicator gene. If the indicator gene is a gene described in i) or ii i) above, the subject is determined to be psoriasis if the expression level is higher than that of the control. If the indicator gene is a gene described in ii) or iv) above, the subject is determined to be psoriasis if the expression level is lower than that of the control.
  • a standard value is usually set based on the expression level of the indicator gene in a healthy person, for example. Based on this standard value, for example, the range of ⁇ 2 S.D. Techniques for setting a standard value and an allowable range based on a measured value of an indicator gene are known.
  • the standard value of the rash-free area in the patient can be determined by measuring the expression level of the indicator gene in the rash-free area in advance. After setting the standard value, only the expression level in the skin area is measured, and the detection method of the present invention is performed based on a comparison with a predetermined standard value in the rash-free area of the patient. You can also.
  • the indicator gene in the subject is the gene described in a) or c) above, the subject is considered to have atopic dermatitis if the expression level is higher than the allowable range compared to the control. Is determined. Similarly, if the indicator gene in the subject is b) or In the case of the gene described in d), the subject is judged to have atopic dermatitis if the expression level is lower than the allowable range compared to the control. If the expression level of the indicator gene is within the acceptable range, the likelihood of atopic dermatitis is expected to be low. '
  • the subject is determined to have psoriasis if the expression level is higher than the allowable range compared to the control. You. Similarly, when the indicator gene in the subject is the gene described in ii) or iv) above, if the expression level is lower than the allowable range compared to the control, the subject will have psoriasis. Is determined. If the expression level of the indicator gene is within the acceptable range, the likelihood of psoriasis is expected to be low.
  • the expression level of the indicator gene includes the transcription of the indicator gene into mRNA and the translation into protein. Therefore, the method for testing atopic dermatitis according to the present invention is performed based on the comparison of the expression intensity of mRM corresponding to the indicator gene or the expression level of the protein encoded by the indicator gene.
  • the measurement of the expression level of the indicator gene in the examination of atopic dermatitis or psoriasis in the present invention can be performed according to a known gene analysis method. Specifically, for example, a hybridization technique using a nucleic acid that hybridizes to the gene as a probe or a gene amplification technique using a DNA that hybridizes to the indicator gene of the present invention as a primer can be used.
  • the probe or primer used for the test of the present invention can be designed based on the base sequence of the indicator gene.
  • the nucleotide sequence of the indicator gene and a part of the amino acid sequence encoded by the indicator gene are known.
  • the GenBank accession numbers of the known nucleotide sequences of the indicator genes of the present invention are as follows: Data 1, Data 2, Data 5, and Data 6 (human), Data 3, Data 4, Data 11, and Data 12 ( Mouse).
  • the nucleotide sequence of a psoriasis indicator gene in the psoriasis test method of the present invention, and A part of the amino acid sequence encoded by this is known.
  • GenBank accession number of the known nucleotide sequence of each indicator gene of the present invention is as described in the following data 7 to data 10 (human).
  • the nucleotide sequence of a part of the indicator gene of the present invention and the amino acid sequence encoded by the nucleotide sequence are shown in the following SEQ ID NOs.
  • SEQ ID NOs amino acid sequence encoded by the nucleotide sequence
  • the indicator gene includes not only human but also homologs of other species. Therefore, an indicator gene in a species other than human refers to a homologue of an indicator gene specific to the species or an exogenous indicator gene introduced into the individual, unless otherwise specified.
  • the homologue of the human indicator gene refers to a gene derived from a species other than human that can be hybridized under stringent conditions using the human indicator gene as a probe.
  • Stringent conditions generally indicate the following conditions. That is, hybridization was carried out at 4 ⁇ SSC at 65 ° C., and 65 was obtained using 0.1 ⁇ SSC. Wash with C for 1 hour.
  • the temperature conditions of the hydripride-wash which greatly affect the stringency, can be adjusted according to the melting temperature (Tm). Tm varies depending on the ratio of constituent bases to the base pairs to be hybridized and the composition of the hybridization solution (salt concentration, sodium formamide / dodecyl sulfate concentration).
  • RNA complementary strand
  • the “complementary strand” refers to one strand of a double-stranded DNA composed of A: T (U in the case of RNA),: C base pair and the other strand.
  • complementary is not limited to ⁇ which is a sequence completely complementary to at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably Should have a homology of 95% or more on the base sequence.
  • the homology of the nucleotide sequences can be determined by an algorithm such as BLAST.
  • Such a polynucleotide can be used as a probe for detecting the indicator gene and as a primer for amplifying the indicator gene.
  • a primer When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp.
  • a DNA having at least a part or all of the sequence of the indicator gene (or its complementary chain) and having a chain length of at least 15 bp is used.
  • the 3'-side region must be complementary, but a restriction enzyme recognition sequence, a tag, or the like can be added to the 5'-side.
  • the “polynucleotide” in the present invention can be DNA or RNA. These polynucleotides may be synthetic or natural.
  • the probe DNA used for hybridization is usually labeled. As the labeling method, for example, the following method can be shown.
  • the term "oligo 'nucleotide” means a polynucleotide having a relatively low degree of polymerization. Oligonucleotides are included in polynucleotides.
  • Inspection of atopic dermatitis using the hybridization technique can be performed using, for example, a Northern hybridization method, a dot plot method, a method using a DNA microarray, and the like.
  • gene amplification techniques such as the RT-PCR method can be used.
  • the expression of the indicator gene of the present invention can be analyzed more quantitatively by using the PCR amplification monitoring method in the gene amplification process.
  • probes that are labeled at both ends with different fluorescent dyes that cancel each other's fluorescence are used to hybridize to the detection target (DNA or RNA reverse transcript).
  • the detection target DNA or RNA reverse transcript.
  • the two fluorochromes separate and the fluorescence is detected. This fluorescence is detected in real time.
  • the number of copies of the target in the target sample is determined based on the number of linear cycles of PCR amplification by simultaneously measuring a standard sample with a clear copy number for the target (Holland, PM et al., 1991, Proc. Natl. Acad. Sci.
  • the method for detecting atopic dermatitis or psoriasis of the present invention comprises It can also be performed by detecting the protein to be loaded.
  • a test method for example, a stamp lotting method, an immunoprecipitation method, an ELISA method, or the like using an antibody that binds to each indicator protein can be used.
  • Antibodies that bind to the indicator protein used for this detection can be obtained using techniques well known to those skilled in the art.
  • the antibody used in the present invention can be a polyclonal antibody or a monoclonal antibody (Milstein C, et al., 1983, Nature 305 (5934): 537-40).
  • a polyclonal antibody against the indicator protein is obtained by removing the blood of a mammal sensitized with the antigen and separating serum from the blood by a known method.
  • serum containing the polyclonal antibody can be used.
  • a fraction containing the polyclonal antibody can be further isolated from the serum.
  • immune cells are removed from a mammal sensitized with the above antigen, and are fused with myeloma cells or the like.
  • the hybridoma thus obtained can be cloned, and the antibody can be recovered from the culture to obtain a monoclonal antibody.
  • antibodies may be appropriately labeled and used for detection of the indicator protein. Further, without labeling the antibody, a substance that specifically binds to the antibody, for example, protein A or protein G can be labeled and detected indirectly. As a specific detection method, for example, an ELISA method can be mentioned.
  • a protein or a partial peptide thereof used as an antigen can be obtained by, for example, incorporating an indicator gene or a part thereof into an expression vector, introducing this into an appropriate host cell, preparing a transformant, and culturing the transformant.
  • the recombinant protein can be obtained by expressing the recombinant protein and purifying the expressed recombinant protein from a culture or a culture supernatant.
  • an oligopeptide consisting of an amino acid sequence encoded by the gene or a partial amino acid sequence of an amino acid sequence encoded by a full-length cDNA can be chemically synthesized and used as an immunogen.
  • the expression level of the indicator gene but also the A test for allergic monopathy or psoriasis can also be performed using the activity of the indicator protein as an indicator.
  • the activity of the indicator protein refers to the biological activity of the protein.
  • a general method for measuring the activity of each protein will be described.
  • Electrophoresis is performed under non-reducing conditions on a SDS polyacrylamide gel obtained by copolymerizing a protease inhibitor with a protease substrate such as gelatin.After the electrophoresis, the gel is incubated at 37 ° C and 16 ° C in an optimal buffer containing protease. Let stand for a while. Sixteen hours later, the gel is stained with Coomassie brilliant blue R250, and the protease inhibitor migration activity 14 can be evaluated based on the fact that the migration position of the protease inhibitor is stained, that is, the gelatin is not degraded.
  • the transcription factor is incubated with a double-stranded oligo DNA containing the target sequence of the transcription factor labeled with 32 P or the like at room temperature to bind. After the incubation, the sample is subjected to electrophoresis on a native polyacrylamide gel containing no SDS, and the mobility of the labeled oligo DNA is evaluated using 32 P radioactivity as an index. If the transcription factor has a binding activity to the oligo DNA, the mobility of the labeled oligo DNA becomes slow and shifts to a higher molecular weight side. Binding specificity for the target sequence is confirmed by an excess of unlabeled double-stranded oligo DNA inhibiting the binding of transcription factor to labeled oligo DNA it can.
  • the ability of the transcription factor to activate the transcription factor can be evaluated by co-transformation of a reporter gene expression vector and a transcription factor expression vector.
  • a reporter gene expression vector is an expression vector in which a reporter gene such as chloramphenicol acetyltransferase (CAT) is linked downstream of its target sequence.
  • the transcription factor whose activity is to be evaluated can be expressed by a transcription factor expression vector in which the transcription factor gene is linked downstream, such as the human cytomegalovirus (CMV) response gene promoter.
  • CMV human cytomegalovirus
  • the transactivity can be evaluated by transgene-introducing these betaters into cell lines such as Hela and HEK293, preparing a cell lysate 48 hours later, and examining the expression level of CAT.
  • myelin basic protein kinase buffer containing as a substrate (20 mM HEPES, pH7 5, 10 raM MgCl 2, 2 mM MnCl 2, 2 mM dithiothreitol, and 25 M ATP.)
  • myelin basic protein kinase buffer containing as a substrate (20 mM HEPES, pH7 5, 10 raM MgCl 2, 2 mM MnCl 2, 2 mM dithiothreitol, and 25 M ATP.
  • To further [ ⁇ - 32 ⁇ ] Add ⁇ and incubate at 37 ° C for 10 minutes. After 10 minutes, stop the reaction with Laeimli buffer, subject the reaction solution to SDS polyacrylamide gel electrophoresis, dry the gel after electrophoresis, and detect the radioactivity of phosphorylated myelin basic protein on an X-ray film.
  • phosphatase to a buffer (25 mM MES, pH 5.5, 1.6 mM dithiothreitol, 10 mM pNPP) containing p-nitrophenyl phosphate (pNPP) as a substrate. Incubate at C for 30 minutes. After 30 minutes, the reaction is stopped by adding IN NaOH, and the absorbance at 405 nm resulting from the hydrolysis of pNPP is measured.
  • a buffer 25 mM MES, pH 5.5, 1.6 mM dithiothreitol, 10 mM pNPP
  • pNPP p-nitrophenyl phosphate
  • [Chemokine, chemokine receptor] The cells in which the chemokine receptor is forcibly expressed are suspended in Hank's balanced salt solution containing the calcium-sensitive fluorescent dye flra-2, and stimulated with a chemokine. The increase in intracellular calcium concentration caused by chemokine stimulation is measured with a fluorescence detector such as LS 50B (PerkinElmer).
  • Cells that express the cytokine receptor are stimulated with the cytokine, and the cell proliferation caused by this is evaluated by thymidine incorporation.
  • the activity of a transcription factor located downstream of a cytokine receptor upon cytokine stimulation can be evaluated by expression of a reporter gene such as luciferase.
  • Cells expressing the adhesion molecule on the cell surface are incubated on a ligand-coated plate, and the number of adhered cells is evaluated.
  • extracellular matrix protein activity To a plate coated with extracellular matrix protein, add a suspension of cells having an extracellular matrix protein receptor such as integrin, and incubate at 37 ° C for 1 hour. After culturing, fix the cells, and add a DNA-binding fluorescent dye such as Hoechst 33342 to react. After the reaction, measure the fluorescence intensity using a fluorimeter. The number of adherent cells quantified as strength is assessed as extracellular matrix protein activity.
  • extracellular matrix protein receptor such as integrin
  • the skin tissue of the subject is used as a sample. Taking a skin tissue sample is somewhat painful to the subject. On the other hand, since skin tissue can be easily collected, it is useful as a diagnostic material.
  • Skin tissue can be collected, for example, as follows. That is, the sampling site is first anesthetized with a local anesthetic. After pulling the skin around the biopsy site to make it slack-free, embed the punch in the skin and rotate to insert the tissue of the specimen into the punch. Pull out the punch and collect the cut skin inside the punch.
  • a punch is a hollow skin tissue sampling device. For example, instruments that can collect skin tissue with a diameter of 3 thighs are commonly used.
  • the skin tissue anatomically includes the epidermis and the dermis.
  • Skin tissue may include non-skin cells found in skin tissue, such as lymphocytes, Langerhans cells, or mast cells, as well as cells specific to the skin. Cells collected together with these skin cells are also included in the skin tissue sample.
  • a skin tissue sample from the rash is used to determine the expression level of the indicator gene in the rash.
  • the rash is the skin that forms the acute lesion.
  • acute lesions can be determined according to the diagnostic criteria reported in Japanese Dermatological Association 104: 1210 (1994). Specifically, for example, the following clinical findings are used as indicators of acute lesions.
  • Acute lesions erythema, wet erythema, papules, serous papules, scales, crusts
  • a skin tissue sample from the rash-free area is used.
  • the rash-free area in a patient is the skin at a site not accompanied by the above lesion.
  • determining the expression level of the indicator gene in the skin of healthy subjects To do this, the skin tissue of a healthy person is used.
  • the healthy subject in the present invention refers to a human who clearly has no disease to be diagnosed. That is, in a test for an allergic disease, a human having no allergic disease is a healthy person. Similarly, in the test method for psoriasis, a person without psoriasis is a healthy subject. A healthy person is allowed to have a disease other than the disease to be diagnosed.
  • a preferred healthy person is a human who has neither allergic disease nor psoriasis.
  • the skin tissue can be collected in the same manner as the patient's skin tissue.
  • skin from different sites can be used for comparison.
  • the collection of skin tissue samples is easy, simple examinations in medical settings are possible. For example, it can be prepared by the method shown in Examples. If the prepared skin tissue is destroyed to form a lysate, it can be used as a sample for immunological measurement of the indicator protein.
  • a lysate is prepared from the above biological sample, it can be used as a sample for immunological measurement of the indicator protein.
  • mRNA is extracted from this lysate, it can be used as a sample for measuring mRNA corresponding to the indicator gene.
  • the indicator protein is secreted into the blood, the expression level of the gene encoding it can be compared by measuring the amount of the target protein contained in a body fluid sample such as blood or serum of the subject. Is possible.
  • the above sample can be used in the method of the present invention after being diluted with a buffer or the like, if necessary.
  • the measured value of the expression level of the indicator gene in the present invention can be corrected by a known method. With the correction, changes in the expression level of the gene in the cells can be compared. Correction of the measured value is performed on the above biological sample.
  • the measurement is performed by correcting the measured value of the expression level of the indicator gene in the present invention based on the measured value of the expression level of a gene whose expression level does not fluctuate greatly (eg, a house-kiving gene). Examples of genes whose expression level does not fluctuate significantly include ⁇ -actin, GAPDH and the like.
  • the present invention provides a reagent for the test method of the present invention.
  • the present invention relates to a test for atopic dermatitis comprising a polynucleotide containing the nucleotide sequence of an indicator gene, or an oligonucleotide having a nucleotide sequence that is complementary to its complementary strand and having a length of at least 15 bases.
  • a reagent for testing atopic dermatitis comprising an antibody that recognizes an indicator protein.
  • the oligonucleotide-antibody constituting the reagent of the present invention can be bound with an appropriate label according to the Atsey format.
  • the oligonucleotide or antibody constituting the reagent of the present invention may be immobilized on a suitable support depending on the assay format.
  • the reagent of the present invention can be used as a test kit by combining the oligonucleotide or the antibody with an additional element necessary for test or storage. Additional components that can make up the kit are shown below. These components can be pre-mixed if necessary. Preservatives and preservatives can be added to each component as needed.
  • the AD index gene in the present invention is expressed in each skin tissue in a rash-free area of the same patient as the eruption of atopic dermatitis patient or in a comparison between a rash-free area of atopic dermatitis patient and a healthy subject Fluctuations in the amount were confirmed. Therefore, the AD indicator gene Atopic dermatitis can be detected using the expression level as an index.
  • the test for atopic dermatitis in the present invention includes, for example, the following tests. Even if a patient cannot demonstrate atopic dermatitis by a general test while showing symptoms suspected of having atopic dermatitis, the test based on the present invention can be considered as a patient with atopic dermatitis. Can be easily determined. More specifically, if the AD indicator gene is a gene described in a) or in patients with symptoms that are suspected of having atopic dermatitis, an increase in the expression of the AD indicator gene indicates the cause of the symptom. Indicates that there is a high possibility of atopic dermatitis.
  • the AD indicator gene is a gene described in b) or d) in a patient with symptoms suspected of having atopic dermatitis
  • a decrease in the expression of the AD indicator gene is attributed to the same atopic cause. This indicates that the possibility of atopic dermatitis is high.
  • tests could be done to determine if atopic '14 dermatitis is improving. In other words, it is useful for judging the therapeutic effect on atopic dermatitis.
  • the AD indicator gene when the AD indicator gene is a gene described in a) or c), the increase in the expression of the AD indicator gene is caused by: a. It is likely that you are doing.
  • the AD indicator gene when the AD indicator gene is a gene described in b) or d), the decrease in the expression of the AD indicator gene is similar to that in atopic dermatitis, Is likely to be
  • the severity of atopic dermatitis can be determined based on the difference in expression level. That is, when the AD indicator gene is a gene described in a) or c), the degree of increase in the expression of the AD indicator gene correlates with the severity of atopic dermatitis. Alternatively, when the AD indicator gene is a gene described in b) or d), the degree of decrease in the expression of the AD indicator gene correlates with the severity of atopic dermatitis.
  • the psoriasis indicator gene according to the present invention is expressed in each skin tissue in an eruption area of the same patient as the skin area of a psoriasis patient, or in a comparison between an eruption area of a psoriasis patient and a healthy person. As a result, a change in the expression level was confirmed. Therefore, psoriasis can be detected using the expression level of the psoriasis indicator gene as an indicator.
  • the psoriasis test in the present invention includes, for example, the following tests. Even if a patient cannot be judged as psoriasis by a general test while showing symptoms suspected of psoriasis, the test according to the present invention can easily determine whether or not the patient is a psoriasis patient. . More specifically, if the psoriatic indicator gene is a gene described in i) or iii) in a patient exhibiting a symptom suspected of psoriasis, an increase in the expression of the psoriasis indicator gene is attributed to psoriasis.
  • the psoriatic index gene is a gene described in ii) or iv) ⁇ , a decrease in the expression of the psoriasis index gene is due to the same psoriasis It indicates that the possibility is high.
  • tests can be done to determine if psoriasis is improving. In other words, it is useful for determining the therapeutic effect on psoriasis. Also, in patients diagnosed with psoriasis, if the psoriasis indicator gene is a gene described in i) or iii), an increase in the expression of the psoriasis indicator gene indicates that psoriasis is more likely to have progressed further. Is shown.
  • psoriasis indicator gene is the gene described in ii) or iv
  • a decrease in the expression of the psoriasis indicator gene indicates that psoriasis is more likely to progress further. Is shown.
  • the severity of psoriasis can be determined based on differences in expression levels. That is, when the psoriasis indicator gene is a gene described in i) or iii), the degree of increase in the expression of the psoriasis indicator gene correlates with the severity of psoriasis. Alternatively, when the psoriasis indicator gene is a gene described in ii) or iv), the degree of decrease in expression of the psoriasis indicator gene correlates with the severity of psoriasis.
  • this endeavor is intended for atopic dermatitis consisting of a transgenic non-human animal with an increased expression level in the skin of the AD indicator gene described in a) or c) or a gene functionally equivalent to the AD indicator gene.
  • atopic dermatitis consisting of a transgenic non-human animal with an increased expression level in the skin of the AD indicator gene described in a) or c) or a gene functionally equivalent to the AD indicator gene.
  • model animals According to the present invention, it has been clarified that the expression intensity of the AD indicator gene described in a) is increased in the rash area of a patient with atopic dermatitis. Similarly, it was clarified that the expression intensity of the AD indicator gene described in c) was increased in a rash-free area of atopic dermatitis patients. Therefore, animals that artificially enhance the expression level of the AD indicator gene described in a) or c) or a gene functionally equivalent to the AD indicator gene in the skin are used as animal models for atopic dermatiti
  • the present invention also relates to an atopic dermatitis comprising a transgenic non-human animal having a reduced expression level in the skin of the AD indicator gene described in b) or d) or a gene functionally equivalent to the AD indicator gene.
  • atopic dermatitis comprising a transgenic non-human animal having a reduced expression level in the skin of the AD indicator gene described in b) or d) or a gene functionally equivalent to the AD indicator gene.
  • the expression intensity of the AD indicator gene described in b) is reduced in the rash of a patient with atopic dermatitis.
  • the expression intensity of the AD indicator gene described in d) was reduced in the eruption-free part of patients with acute dermatitis. Therefore, animals that artificially reduce the expression level of the AD indicator gene described in b) or d) or a gene functionally equivalent to the indicator gene in the skin are used as animal models for atopic dermatitis be able to.
  • the present invention relates to a psoriatic model animal comprising a transgenic non-human animal having an increased expression level in the skin of the psoriasis indicator gene described in i) or iii) or a gene functionally equivalent to the psoriasis indicator gene.
  • the expression intensity of the psoriasis indicator gene described in i) increases in the rash of psoriatic patients.
  • the expression intensity of the psoriasis indicator gene described in iii) was increased in the rash-free area of psoriatic patients. Therefore, an animal that artificially enhances the expression level of the psoriasis indicator gene described in i) or iii) or a gene functionally equivalent to the psoriasis indicator gene in the skin is a psoriatic model animal. Can be used.
  • the present invention provides a transjeet with reduced expression intensity in skin of a psoriasis indicator gene or a gene functionally equivalent to the psoriasis indicator gene described in ii) or iv).
  • the present invention relates to a psoriatic model animal comprising a nonhuman animal.
  • the expression intensity of the psoriasis indicator gene described in ii) is reduced in the rash of psoriatic patients.
  • the expression intensity of the psoriasis indicator gene described in iv) was reduced in the rash-free area of psoriatic patients. Therefore, an animal whose expression level of the psoriasis indicator gene described in ii) or iv) or a gene functionally equivalent to the psoriasis indicator gene in the skin is artificially reduced is used as a psoriasis model animal. can do.
  • a functionally equivalent gene is a gene that encodes a protein having an activity similar to the activity clarified in the protein encoded by the indicator gene.
  • a representative example of a functionally equivalent gene is the power gene partner of the indicator gene in the animal species that the test animal originally has.
  • the genes described in the above groups A) to D) are functionally equivalent genes in mice.
  • the genes described in the groups A) to D) are desirable indicator genes when the screening according to the present invention is performed using mice.
  • mice of the AD indicator gene described in a) to d have clarified the counterpart in mice of the AD indicator gene described in a) to d).
  • the mouse interaction percentages against the AD indicator gene described in a) -d) are described as A) -D), respectively.
  • These counterparts are genes that showed a two-fold or more difference when comparing the expression levels of the genes between the sensitized mouse auricle skin and the non-sensitized mouse auricle skin.
  • an atopic dermatitis model animal can be created by adjusting the expression level of these counterparts or by administration. That is, the present invention relates to a method for producing an animal model for atopic dermatitis by regulating the expression levels of the genes described in A) to D). Alternatively, the present invention relates to a method for producing an atopic dermatitis model animal, comprising a step of administering a protein itself encoded by the genes described in A) to D) or an antibody of the protein to a non-human animal.
  • atopic dermatitis can be induced by increasing the expression level.
  • atopic dermatitis model animals can be created by administration of genes selected from these gene groups and the proteins encoded by the genes. Since all of these counterparts are mouse genes, it is desirable to administer genes and proteins to mice when administering them.
  • atopic dermatitis can be induced by suppressing the expression level of the gene group described in ii) or D), similarly to the gene group described in b) or d).
  • atopic dermatitis can be created by suppressing the expression of a gene selected from these genes and the activity of the protein encoded by the gene.
  • Antisense nucleic acid or RNAi can be used to suppress expression.
  • Administration of an activity inhibitor such as an antibody is effective for regulating the activity of a protein. That is, atopic dermatitis is induced by administering these components to an animal having the gene group described in B) or D), ie, a mouse, innately.
  • the atopic dermatitis model animal is useful for clarifying in vivo changes in atopic dermatitis. Furthermore, using the atopic dermatitis model animal to elucidate further functions of the indicator gene and evaluating a drug targeting the gene are of great significance.
  • the atopic dermatitis model animal according to this effort is useful for elucidating the mechanism of atopic dermatitis and for testing the safety of screened compounds.
  • an animal model of atopic dermatitis according to the present invention develops dermatitis or shows a change in a measurement value associated with any allergic disease
  • a screening system for searching for a compound having an effect of restoring it is shown. Can be constructed.
  • an increase in the expression level refers to a state in which the indicator gene has been introduced as a foreign gene and is forcibly expressed, or a state in which the transcription and translation of the indicator gene originally provided in the test animal are enhanced.
  • Degradation of proteins that are translation products Means any of the suppressed states.
  • a decrease in the expression level means either a state in which transcription of an indicator gene provided in a test animal and translation into a protein are inhibited, or a state in which degradation of a protein as a translation product is promoted.
  • the gene expression level can be confirmed, for example, by the difference in signal intensity on a DNA chip as shown in the Examples.
  • the activity of the protein as a translation product can be confirmed by comparison with a normal state.
  • transgenic animals include animals into which the indicator gene has been introduced and forced to be expressed, animals in which the indicator gene has been knocked out, and animals in which the gene has been replaced (knocked in) with other genes.
  • transgenic animals into which antisense DNA against the indicator gene, DNA encoding lipozyme, DNA functioning as a decoy nucleic acid, or the like has been introduced can also be used as the transgenic animal in the present invention.
  • animals in which a mutation is introduced into the coding region of the indicator gene to enhance or suppress its activity, or in which an amino acid sequence which is hardly degraded or easily degraded can be shown.
  • the mutation in the amino acid sequence can be substitution, deletion, insertion, or addition.
  • the expression itself of the indicator gene of the present invention can be regulated by mutating the transcriptional regulatory region of the gene.
  • transgenic animals for specific genes are known. That is, a method in which a gene and an egg are mixed and treated with calcium phosphate, and a method in which a gene is directly introduced into a nucleus of a pronuclear stage egg with a micropipette under a phase-contrast microscope (micro u injection method, US Pat. No. 4,873,191). No.), transgenic animals can be obtained by a method using embryonic stem cells (ES cells).
  • ES cells embryonic stem cells
  • a method has been developed in which a gene is inserted into a retrovirus vector to infect eggs, and a sperm vector method in which a gene is introduced into eggs via sperm.
  • the sperm vector method is to attach a foreign gene to sperm or use a method such as electroporation to control sperm cells. This is a genetic recombination method in which a foreign gene is introduced by fertilizing an egg after it has been incorporated into the egg (M. Lavitranoe et al., Cell, 57, 717, 1989).
  • the expression level of the exogenous indicator gene in the transgenic animal can be adjusted by administering the substance. it can.
  • the transgenic animal used as a model animal for atopic dermatitis or psoriasis of the present invention can be prepared using any vertebrate other than human. Specifically, transgenic animals in which the introduction of various genes and the expression levels of various genes have been modified in vertebrates such as mice, rats, egrets, miniptas, goats, sheep, and magpies have been created.
  • the present invention relates to a method for screening a candidate compound for treating atopic dermatitis.
  • the AD indicator gene is a gene selected from the group described in any of a) to a).
  • the expression level of the gene selected from the group described in a) is significantly increased in the eruption area of the same patient as compared to the eruption area of the atopic dermatitis patient.
  • the expression level of the gene selected from the group described in b) is significantly lower in the rash of the same patient than in the non-rash of the atopic dermatitis patient.
  • the expression level of the genes selected from the group described in c) is significantly increased in the eruptions of atopic dermatitis patients as compared with healthy subjects.
  • Genes selected from the group described in d) have significantly lower expression levels in the eruptions of atopic dermatitis patients than in healthy subjects.
  • a therapeutic agent for atopic dermatitis can be obtained by selecting a compound that can reduce the expression level of the AD indicator gene. it can.
  • a therapeutic agent for atopic dermatitis can be obtained by selecting a compound capable of increasing the expression level of the AD indicator gene.
  • the screening method of the present invention for the treatment of atopic dermatitis drug candidates thus, the following genes can be shown as preferred genes for the AD indicator gene.
  • Preferred genes as a gene of a) group The following GenBank of ⁇ click session number AI6803 50, AI261490, N22028, AI458014 , AI760613, M191741, AI129310, W44526, AWO 14646, AI913548, AI768116, AA652869 S AW005250, AI655668, AA765843, AI6322 23, AI807277, AI763378, AA196201, AI301935, AI431800, AI968085, AI472111 AI088609, AI983204, A orchid 4155, AI718763, AA424160, N32858, AI971000, AI655 719, AI817147, AI986192 ,. , AI446030, H06350, AI671741, AI983994, and any of the genes selected from the genes containing or including the base sequence shown in AI039915
  • AD indicator genes have no change in expression levels in patients with psoriasis. In other words, it can be said that the gene whose expression level has been specifically changed in patients with atopic dermatitis.
  • AD indicator genes it is possible to evaluate the therapeutic effects specific to atopic dermatitis symptoms.
  • the present invention relates to a method for screening a drug combination for treating psoriasis.
  • the psoriasis indicator gene is a gene selected from the group described in any of i :) to iv).
  • the expression level of the gene selected from the group described in i) is significantly increased in the rash area of the same patient compared to the non-rash area of the psoriatic patient.
  • Genes selected from the group described in ii) have significantly lower expression levels in the rash area of the same patient as compared to the non-rash area of the psoriatic patient.
  • the expression level of the gene selected from the group described in iii) is significantly increased in the rash-free area of psoriatic patients as compared with healthy individuals. And the expression level of the gene selected from the group described in iv) is significantly lower in the eruption of psoriatic patients than in healthy individuals.
  • the psoriasis index By selecting a compound that can reduce the expression level of a gene, a therapeutic agent for psoriasis can be obtained.
  • a therapeutic agent for psoriasis can be obtained by selecting a compound capable of increasing the expression level of the psoriasis indicator gene.
  • the following genes can be shown as preferred genes as psoriatic index genes.
  • AI669994, AI440145, AW003577 , AA903403 S AI697887, AI760366, AI799626, AA7 60767, AI567489, AA010318 N AI434862, AI678727, AI829641, AA535978 S AI0171 78, AI703265, AI832016, AI979308, AI344053, AI927079, AI798407, R53734, A 1282982, M789312, AA707213, M535031, AA770302, M932068
  • a compound that increases the expression level of a gene is a compound that has an action that promotes any of the steps of gene transcription, translation, and protein activity expression.
  • the compound that decreases the expression level of a gene is a compound that has an inhibitory action on any of these steps.
  • the method of screening a candidate compound for treating an allergic disease (or psoriasis) of the present invention can be performed in vivo or in vitro. This screening can be performed, for example, according to the following steps.
  • any one of the genes and functions selected from the group described in any of the above a) to d) (any one of i) to iv) for psoriasis) Genes equivalent to each other can be used.
  • a functionally equivalent gene is a gene that encodes a protein having an activity similar to the activity clarified in the protein encoded by the indicator gene.
  • a representative example of a functionally equivalent gene is the power gene partner of the indicator gene in the animal species that the test animal originally has.
  • an atopic dermatitis model animal can be used as a test animal in the screening method of the present invention.
  • Atopic dermatitis model animals are known.
  • a spontaneous dermatitis model using NC / Nga mice has been reported.
  • a total of eight administrations of mite antigen (5 ig / ear) to the pinna of this mouse at 2-3 day intervals can induce symptoms very similar to human atopic dermatitis after 2 weeks.
  • the screening according to the present invention can be performed by administering the candidate conjugate to this system and tracking the change in the expression level of the indicator gene of the present invention.
  • the effect of the drug candidate compound on the expression level of the indicator gene is evaluated by contacting the drug candidate compound with the test animal and monitoring the effect of the compound on the expression of the indicator gene in a biological sample derived from the test animal. can do. Fluctuations in the expression level of the indicator gene in a biological sample derived from a test animal can be monitored by a method similar to the test method of the present invention. Furthermore, based on the results of this evaluation, if the indicator gene is a gene described in a) or c) group (for psoriasis, i) or iii) group, a drug candidate compound that reduces the expression level is identified. If the indicator gene is a gene described in group b) or d) (for psoriasis, group ii) or iv), a drug candidate that increases the expression level is selected. Hou Co-compounds can be screened.
  • the screening according to the present invention is performed by collecting a skin tissue sample from a test animal and comparing the expression level of the indicator gene with a control not contacting the candidate.
  • Methods for collecting and preparing skin tissue samples are known.
  • Such a screen allows selection of drugs that participate in the expression of the indicator gene in various forms. Specifically, for example, drug candidate compounds having the following actions can be found.
  • the indicator gene is a gene described in a) group or c) group (for psoriasis, group i) or iii)):
  • the indicator gene is a gene described in group b) or d) (for psoriasis, group ii) or iv):
  • a candidate compound is brought into contact with a cell that expresses an indicator gene, and the indicator gene is a group or c) group (for psoriasis, i) group or iii) group).
  • the indicator gene is a gene described in group b) or d) (for psoriasis, a group described in group ii or iv)
  • a compound that decreases the expression level A method of selecting a compound that increases the expression level can be mentioned. This screening can be performed, for example, according to the following:!: Procedure.
  • the level of expression of the indicator gene described in a) or c) group (i) or iii) group for psoriasis is lower than that of a control not contacted with the candidate compound. Selecting a compound that increases the expression level of the gene to be expressed in the group b) or d) (group ii) or group i V) in the case of psoriasis.
  • cells that express the indicator gene can be obtained by inserting the indicator gene into an appropriate expression vector and introducing the vector into an appropriate host cell.
  • Usable vectors and host cells may be any as long as they can express the indicator gene of the present invention. Examples of host cells in the host-vector system include Escherichia coli, yeast, insect cells, animal cells, and the like, and any available vector can be appropriately selected.
  • Examples of a method for introducing a vector into a host include a biological method, a physical method, and a chemical method.
  • Biological methods include, for example, a method using a viral vector, a method using a specific receptor, a cell fusion method (HVJ (Sendai virus), polyethylene glycol (PEG), an electric cell fusion method, micronucleus fusion). Method (transfer of chromosomes)).
  • HVJ Sendai virus
  • PEG polyethylene glycol
  • Method transfer of chromosomes
  • Method transfer of chromosomes
  • microinjection comb Yung method, electroporation Chillon method, and a method of have use Gene particle gun (gene gun).
  • Chemical methods include the calcium phosphate precipitation method, liposome method, DEAE dextran method, protoplast method, erythrocyte ghost method, erythrocyte membrane ghost method, and microcapsenole method.
  • skin tissues such as Langerhans cells, mast cells, T cells, eosinophils, B cells, neutrophils, or basophils are used as the cells expressing the indicator gene.
  • skin tissues such as Langerhans cells, mast cells, T cells, eosinophils, B cells, neutrophils, or basophils are used as the cells expressing the indicator gene.
  • keratinocytes Primary culture of human keratinocytes (keratinocytes) stimulates caloric stimulation of NHEK (Normal Human Epidermal Keratinocyte) with TGF-] 3 or sodium butyrate It has been reported that the ability to derive a fraction can be obtained by reading (eg, Geng Wang et al., EXPERIMENTAL CELL RESEARCH 198, 27-30 (1992)).
  • An intracellular structure called cornified envelope (CE) is formed during differentiation.
  • the screening can be confirmed using the formation of CE or the gene expression of CE constituent molecules (involucrin, loricrin, etc.) as an index.
  • the cells thus separated are useful for screening in the present invention.
  • Skin cells, T cells, eosinophils, mast cells, basophils, B cells, Langerhans cells, and neutrophils can also be used as cells found in skin tissue.
  • the strain E. coli skin cells are suitable for the screening method of the present invention because homogeneous cells can be obtained in large quantities and cultivation is easy.
  • the following are examples of skin cell lines that can be used in the present invention.
  • T cell lines Jurkat (ATCC TIB-152), Molt-4 (ATCC CRL-1582), H9 (ATCC HTB-176)
  • One cell line B cell DND39, Raji (ATCC CCL-86)
  • a candidate compound is brought into contact with the established skin cells. Then, the expression level of the indicator gene in the skin cells of the strain I was measured, and compared with a control not contacted with the candidate compound, a) group or c) group (for psoriasis, i) group or ii).
  • a compound that decreases the expression level of the gene is used, and in the b) group or d) group (for psoriasis, the ii) group or iv) group is used.
  • a compound that increases the expression level of the gene is selected.
  • the expression level of the indicator gene can be compared not only with the expression level of the protein encoded by the gene, but also by detecting the corresponding mRNA.
  • the step of preparing an mRNA sample as described above is performed instead of the step of preparing a protein sample. Detection of mRNA and protein can be carried out by known methods as described above.
  • a transcriptional regulatory region of the indicator gene of the present invention can be obtained, and a reporter Atssei system can be constructed.
  • the reporter atsey system refers to an atsey system that screens for a transcriptional regulatory factor acting on the transcriptional regulatory region using the expression level of a reporter gene located downstream of the transcriptional regulatory region as an index.
  • the present invention provides a method for screening a therapeutic agent for atopic dermatitis or psoriasis, which comprises the following steps, wherein the indicator gene is any one of a) to (for psoriasis, any one of i) to iv) A) a gene selected from the group described in (1), or a gene functionally equivalent to the indicator gene.
  • Transcription regulatory regions include promoters, enhancers, and usually promoters. For example, CMT boxes, TATA boxes, and the like found in one region can be exemplified. Reporter: As the offspring, CAT ⁇ chloramphenicol acetyl transferase) m. Gene, luciferase (lucif erase) gene, growth hormone gene, etc. can be used.
  • the transcription regulatory region of each indicator gene in the present invention can be obtained as follows. That is, first, based on the nucleotide sequence of the cDNA disclosed in the present invention, screening is performed from human genomic DNA libraries such as BAC library and YAC library by a method using PCR or hybridization, and a genome containing the cDNA sequence is used. Obtain a DNA clone. Based on the sequence of the obtained genomic DNA, the transcription control region of the cDNA disclosed in the present invention is estimated, and the transcription control region is obtained. The obtained transcription regulatory region is cloned so as to be located upstream of the reporter gene to construct a reporter construct. The resulting reporter construct is introduced into a cultured cell line to obtain a transformant for screening.
  • the transformant was contacted with a candidate compound, and compared to a control not contacted with the candidate compound, the index gene described in group a) or c ) (group i) or iii) for psoriasis)
  • a compound that reduces the expression level of the reporter gene, and an indicator gene described in group b) or d) (group ii) or iv) for psoriasis) the expression level of the reporter gene is reduced.
  • the screening of the present invention can be performed by selecting a compound to be increased.
  • the present invention provides a method for screening a therapeutic agent for atopic dermatitis or psoriasis, comprising the following steps, wherein the indicator gene is any one of a;) to d) (i) to iv for psoriasis.
  • the present invention relates to a method which is any gene selected from the group described in any one of the above) or a gene functionally equivalent to the indicator gene.
  • the activity of the indicator protein of the present invention as an indicator, for the indicator genes described in a) or c) (in the case of psoriasis, the i) or iii) group, compounds having an activity of inhibiting the activity Can be screened.
  • the compound thus obtained suppresses the function of each of the indicator genes described in a) or c) (for psoriasis, i) or iii)).
  • atopic dermatitis (or psoriasis) can be controlled through inhibition of the indicator protein whose expression is induced in the skin.
  • the indicator genes described in b) or d) for psoriasis, ii) or iv
  • compounds having an activity to promote the activity can be screened.
  • the compounds obtained in this way promote the function of the indicator genes described in groups b) or d) (groups ii) or iv) for psoriasis.
  • atopic dermatitis or psoriasis
  • test candidate substances used in these screenings include compound preparations synthesized by existing chemical methods such as steroid derivatives, compound preparations synthesized by combinatorial chemistry, extracts of animal and plant tissues, or microorganism cultures.
  • a mixture containing a plurality of compounds such as a product, a sample purified from the mixture, and the like.
  • -Polynucleotides, antibodies, cell lines, or model animals required for various screening methods according to the present invention can be combined in advance to form a kit.
  • kits include substrate compounds used to detect the label, Media and containers, positive and negative standard samples, and instructions describing how to use the kit can also be packaged.
  • the compound selected by the screening method of the present invention is useful as a therapeutic agent for atopic dermatitis.
  • antisense DNA capable of suppressing the expression of any of the AD indicator genes described in a) and c) is also useful as a therapeutic agent for atopic dermatitis.
  • an antibody that recognizes a protein encoded by any of the AD indicator genes described in a) or c) is also useful as a therapeutic agent for atopic dermatitis.
  • the AD indicator gene described in a) or c) is a gene whose expression is increased in the rash area of atopic dermatitis patients. Therefore, a therapeutic effect on atopic dermatitis can be expected by suppressing the expression of these genes or the functions of the proteins encoded by these genes.
  • AD indicator genes described in b) or d), as well as the protein itself encoded by the gene are also useful as therapeutic agents for atopic dermatitis. '
  • the compound selected by the screening method of the present invention is useful as a therapeutic agent for psoriasis.
  • an antisense DNA capable of suppressing the expression of any of the psoriasis indicator genes described in i) and iii) is also useful as a therapeutic agent for psoriasis.
  • an antibody that recognizes a protein encoded by any one of the psoriasis indicator genes described in i) or iii) is also useful as a therapeutic agent for psoriasis.
  • the indicator gene described in i) or iii) is a gene whose expression increases in the rash of psoriasis patients. Therefore, a therapeutic effect on psoriasis can be expected by suppressing the expression of these genes or the functions of the proteins encoded by these genes.
  • any psoriasis indicator gene described in ii) or iv), and the gene The protein itself is also useful as a therapeutic agent for psoriasis.
  • the therapeutic agent for allergic disease or psoriasis of the present invention comprises a compound selected by the screening method as an active ingredient, and is mixed with a physiologically acceptable carrier, excipient, diluent, or the like. Can be manufactured.
  • the therapeutic agent for allergic disease or psoriasis of the present invention can be administered orally or parenterally for the purpose of ameliorating allergic symptoms or psoriasis.
  • dosage forms such as granules, powders, tablets, capsules, solvents, emulsions, and suspensions can be selected.
  • Injections include subcutaneous injections, intramuscular injections, and intraperitoneal injections.
  • a therapeutic effect can be achieved by introducing a gene encoding the protein into a living body using a gene therapy technique.
  • Techniques for treating a disease by introducing a gene encoding a protein having a therapeutic effect into a living body and expressing the gene are known.
  • antisense DNA can be incorporated downstream of an appropriate promoter sequence and administered as an antisense RNA expression vector.
  • this expression vector is introduced into mononuclear cells of an allergic disease patient, the antisense of these genes is expressed, and a therapeutic effect of allergic disease can be achieved by reducing the expression level of the genes.
  • Methods for introducing an expression vector into mononuclear cells are known in vivo and in vivo.
  • the dosage varies depending on the age, sex, weight and condition of the patient, therapeutic effect, administration method, processing time, or the type of active ingredient contained in the pharmaceutical composition, but is usually once per adult.
  • the present invention provides at least one type of AD finger selected from any of the groups a) to d).
  • the present invention provides a DNA chip for diagnosing psoriasis, on which a probe for measuring at least one type of psoriasis indicator gene selected from any of groups i) to iv) is immobilized.
  • the type of indicator gene to be measured is arbitrary. The larger the number of indicator genes, the more judgments can be made based on more indicators. In general, the accuracy of a power diagnosis that measures more indicators increases.
  • it is advantageous to select genes having different properties. Genes that are considered to differ in the mechanism of expression level fluctuation or the function of the protein encoded by the gene can be referred to as genes having mutually different properties.
  • the same DNA chip can be used for both atopic dermatitis and psoriasis. Can also be tested by using the AD indicator gene described in a) -d) and the psoriasis indicator gene described in i) -iv) as the measurement targets, the same DNA chip can be used for both atopic dermatitis and psoriasis. Can also be tested by
  • the following examples can be shown as examples of combinations of indicator genes.
  • the following combinations of indicator genes contribute to the improvement of the accuracy of allergy tests.
  • Proteases and protease inhibitors are indicators of the balance between tissue disintegration and architecture. That is, among the indicator genes of the present invention, for a gene selected from a gene included in a protease group and a gene selected from a gene included in a protease inhibitor, a probe for detecting the gene is integrated, and a chip for detecting atopic dermatitis is obtained. It can be.
  • the indicator genes included in each group can be found from the list of indicator genes shown at the end of the description.
  • any of these combinations is a combination of substances having a relationship of ligand and receptor with each other.
  • the immune response can also be viewed as a result of the interaction between these substances. Therefore, by combining these indicator genes, it may be possible to determine the immunological condition of skin tissue.
  • As the indicator gene both a molecule having a relationship between a ligand and a receptor may be selected, or if any of them is not indicated as an indicator gene in the present invention, only one of them is used. Can also be selected as an indicator gene.
  • extracellular matrix proteins examples include collagen and the like.
  • Cytoskeletal proteins include keratin and small prolin rich protein ⁇ involucrin.
  • intercellular adhesion molecules include cadherin, desmocholine, and the like.
  • the transcription factor can be jun, fos, or myc.
  • the indicator gene contained in the enzyme By selecting the indicator gene contained in the enzyme, it is possible to know what kind of metabolism is performed in skin cells (epidermal keratinocytes and dermal fibroblasts). For example, the metabolism of lipid mediators and lipid molecules that support Paria function can be determined from the expression levels of lipid metabolizing enzymes.
  • Lipid-metabolizing enzymes include, for example, phospho-ipase A2, eye-oxygenase-2, prostagrandin D2 synthase, and Fatty acid desaturase 1,2.
  • detecting the selected gene by combining any plural genes selected from the genes constituting the groups a) and b) An atopic dermatitis test chip that integrates a probe that can be used is effective.
  • group a usually 10 or more, for example, 30 or more, preferably 50 or more, more preferably 60 or more, further preferably 80 or more, or 10.0 or more genes are selected. be able to.
  • group b usually 10 or more, for example, 30 or more, preferably 50 or more, more preferably 60 or more, and still more preferably 80 or more genes can be selected.
  • the c) group or d) group will be detected. Detecting the selected gene for any combination of multiple genes selected from the gene A tip for atopic dermatitis, which integrates a probe that can be used, is effective. Specifically, 10 or more, for example, 30 or more, preferably 50 or more, more preferably 60 or more, and still more preferably 80 or more genes can be selected from the group c). On the other hand, from group d), usually 10 or more, for example, 30 or more, preferably 50 or more, more preferably 60 or more, and still more preferably 70 or more genes can be selected.
  • a chip for detecting atopic dermatitis comprising a combination of a gene selected from the group a) and a gene selected from the group c) is also effective for improving the test accuracy.
  • a chip for atopic dermatitis testing comprising a combination of a gene selected from the group b) and a gene selected from the group d) is also effective for improving the test accuracy.
  • a plurality of indicator genes can be combined in the same manner as the DNA chip for AD diagnosis.
  • a psoriasis detection chip can be obtained by combining the psoriasis indicator genes described in i) to Lv).
  • the psoriasis indicator gene can be combined in the same manner as the atopic dermatitis test chip described above.
  • Example 1 Analysis of differential expression in skin tissue of patients with atopic dermatitis using Affimetrics GeneChip
  • the three collected skin biopsy (diameter 3 bun) were immersed in Isogen (Nippon Gene; Wako Pure Chemical), and homogenized using an Ultratax T8 homogenizer (IKA). After homogenization, total RNA was extracted according to the manual of Isogen. A black-mouthed form was added, and the mixture was centrifuged with stirring to collect an aqueous layer. Next, isopropanol was added and the mixture was centrifuged with stirring to collect the precipitate. The precipitate was rinsed with 75% ethanol and centrifuged, and the precipitate was collected as total RNA. The collected total RNA was further purified using the RNeasy Mini kit (QIAGEN) according to the manual.
  • Isogen Natural Gene
  • IKA Ultratax T8 homogenizer
  • RNA 2 From Total RNA 2—, use T7— (dT) 24 (Amersham Pharmacia Biotech) as a primer, and use Superscript II Reverse Transcriptase (Life Technologies) from Affymetrix Expression Analysis Technical Manual. And reverse transcribed to produce single-stranded cDNA.
  • the T7-(dT) 24 primer consists of a nucleotide sequence obtained by adding (dT) 24 to the nucleotide sequence of the T7 promoter as follows.
  • DNA Ligase, DNA polymerase I and RNase H were added to synthesize double-stranded cDNA. After extracting the cDNA with phenol / cloth form, it was subjected to Phase Lock Gels and purified by ethanol precipitation. Furthermore, biotin-labeled cRNA was synthesized using the BioArray High Yield RNA Transcription Labeling Kit. CRNA was purified using RNeasy Spin column (QIAGEN) and fragmented by heat treatment.
  • Streptavidin-Phycoerythrin was added for staining. After washing, a mixture of normal goat IgG and biotinylated goat anti-streptavidin IgG antibody was placed on the array. Furthermore, in order to enhance the fluorescence intensity, Streptavidin-Phycoerythrin was added again for staining. After washing, it was set on a scanner and analyzed with a DNA chip analysis software.
  • the expression fluorescence intensity was measured using Suite, a DNA chip analysis software, and the data was analyzed. First, Absolute analysis was performed on all chips, and the gene expression ⁇ of each sample used was measured.
  • Gene expression in rash-free areas and acute lesions in 10 patients was compared by Co-immediate arison analysis, and genes whose expression was increased more than 2-fold in acute lesions compared to those in non-rashes and those whose expression was reduced to less than 1/2 were selected.
  • genes that are commonly fluctuating in more than 6 of the 10 patients we now select genes that are commonly fluctuating in more than 6 of the 10 patients. Tables 2 to 6 show the gene names of the selected genes and the expression profiles in each case. These genes, which are commonly fluctuated in several patients with atopic dermatitis, are thought to play an important role in the pathogenesis of atopic dermatitis, and are therefore important as diagnostic markers and therapeutic targets. It is suggested.
  • ⁇ ⁇ ⁇ indicates that the fluctuation is 50 times or more, ⁇ ⁇ 10 is 10 to 50 times, ⁇ 3 is 3 to 10 times, ⁇ is 2-3 times, ⁇ is no change, ⁇ ⁇ indicates 1/50 or less, ⁇ ⁇ 1 indicates 1/50 to 1/10 times, ⁇ ⁇ indicates 1/10 to: 1/3 times, ⁇ indicates 1/3 to 1/2 times .
  • ⁇ ⁇ ⁇ ⁇ ⁇ has a fluctuation of 50 times or more, ⁇ ⁇ 10 10 to 50 times, ⁇ 3 3 to 10 times, ⁇ 2 to 3 times, one does not change, ⁇ i Is 1/50 times or less, ⁇ ⁇ ⁇ is 1/50 ⁇ : 1/10 times, li ft 1/10 ⁇ ; 1/3 times
  • the gene encodes a humoral factor or a membrane protein, a neutralizing antibody, a humoral factor itself, or a soluble receptor can be administered to an experimental animal.
  • a humoral factor or a membrane protein a neutralizing antibody, a humoral factor itself, or a soluble receptor
  • the importance of the gene in the pathogenesis of human dermatitis can be evaluated in a shorter time than when a genetically modified mouse is used.
  • the gene expression profile of a mouse dermatitis model was analyzed, and human atopic dermatitis was analyzed. Compared with atopic dermatitis.
  • Mite antigen (Dermatophagides pteronyssinus / site) was intradermally administered to the auricle and back skin of SPF NC / Nga mice (6 weeks old) 9 times at 3 day intervals in total. Ear edema Measurements and back skin symptoms were observed weekly. The blood total IgE concentration was measured before the administration of the mite antigen, 14 days and 28 days after the start of the administration, and measured using a mouse IgE measurement kit (Yamasa soy sauce). As a control, a non-sensitized group was taken, and the test was carried out using 20 animals (10 animals: dissected 14 days after the start of mite antigen administration, 10 animals: 28 days after the start of mite antigen administration).
  • Ear edema rate (%) Ear thickness after sensitization / Ear thickness before sensitization x 100
  • Non-sensitized group Mean 0 23.7 19.3
  • Sensitization group Mean 0 183 250.4
  • Ear skin and back skin were collected from non-sensitized mice and sensitized mice (14 and 28 days after sensitization), immersed in Isogen (Nippon Gene; Wako Pure Chemical), and homogenized ( Homogenized under ice-cooling using NS-310E; After the homogenization, total RNA was extracted according to the manual of Isogen. Chloroform was added, and the mixture was centrifuged with stirring to collect an aqueous layer. Next, isopropanol was added, and the mixture was centrifuged with stirring to collect the precipitate. The precipitate was rinsed and centrifuged at 75 ° / 0 ethanol, and the precipitate was collected as total RNA. The collected total RNA was further purified using the RNeasy Mini kit (QIAGE N) according to the manual.
  • RNA extracted from the pinna of each individual of 10 animals per group was collected, and the total RNA was 25 ⁇ g force, T7- (dT) 24 (Amersham Pharmacia Biotech) as a primer, and the expression of Aflymetrix was used.
  • reverse cycling was performed using Superscript II Reverse Transcriptase (Life Technologies) to produce single-stranded cDNA.
  • the T7- (dT) 24 primer consists of a nucleotide sequence obtained by adding (dT) 24 to the nucleotide sequence of the T7 promoter as follows.
  • Streptavidin-Phycoerythrin was added for staining. After washing, a mixture of normal goat IgG and a biotinylated goat anti-streptavidin IgG antibody was added to the array. Furthermore, in order to enhance the fluorescence intensity, Streptavidin-Phycoerythrin was added again for staining. After washing, it was set on a scanner and analyzed with a DNA chip analysis software.
  • the expression fluorescence intensity was measured using Suite, a DNA chip analysis software, and the data was analyzed. First of all chips for follow GeneChip Analysis Suite User Guide performs Absolute analysis, to measure the gene expression level of samples each using c
  • Avg Diff Average Difference
  • ⁇ ⁇ ⁇ indicates that the fluctuation is 50 times or more, ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 50 ⁇ ⁇ ⁇ ⁇ ⁇ 1 ⁇ 1 1 Is 1/50 or less, ⁇ i is 1 / 50-; 1 / 10-fold, ⁇ I is 1 / 10-; 1 / 3-fold, ⁇ is 1 / 3-1 / 2-fold.
  • genes were identified as genes that can be used as indicators of atopic dermatitis. Any of these genes can be used as an indicator gene in the present invention.
  • interleukin 1 delta / 84883—at / AI040890 / thigh—012275 / NP-1 036407 / Biochem. Biophys. Res.Commun. 263 (3), 702-706 (1999) //
  • epithelial protein up- regulated in carcinoma, membrane associated protein
  • peptidylprolyl isomerase (cyclophilin) —like 1 51169—at / 524353 / customer—016059 / NP—057143 / Cytogenet.Cell Genet.
  • protease, serine, 3 (mesotrypsin) / 60083_at / AW007273 / M_002771 / P_002762 / Gene 136 (1-2), 167-175 (1993) //
  • centaurin alpha 2 / 44029_at / AI761520 / NM_018404 / NP_060874 / Genomics 66 (1) 93-97 (2000) //
  • solute carrier family 7, (cationic amino acid transporter, y + system) membrane / 54049_at / AI652991 / NM_014331 / NP_055146 / J. Biol. Chem. 274 (17), 11 455-11458 (1999) //
  • solute carrier family 6 neurotransmitter transporter
  • gap junction protein beta 2, 26kD (connexin 26) / 80408_at / AA961504 / M_004 004 / NP—003995 / J. Cell Biol. 118 (5), 1213—1221 (1992) //
  • tumor necrosis factor (ligand) superfamily member 13b / 84765_at / AI446030 / NM_006573 / NP_006564 / J. Leukoc. Biol. 65 (5), 680-683 (1999) //
  • Wiskott-Aldrich syndrome (eczema-thrombocytopenia) / 64315_r_at / AI655719 / NM _000377 / P_000368 / Lancet 1 (7377), 119-123 (1965) //
  • SAM domain, SH3 domain and nuclear localisation signals l / 50026_at / AI823 872 / marauder 022136 / P-071419 / Unpublished (1999) / SEQ ID NO: 12 (base sequence), SEQ ID NO: 130 (amino acid sequence) I
  • epithelial stromal interaction 1 (breast) / 90421_at / AA633203 / N3 ⁇ 4L033255 / P_150280 / Unpublished / SEQ ID NO: 21 (base sequence), SEQ ID NO: 13 9 (amino acid sequence) /
  • FLJ21531 f is, clone COL06036 / 42270— at / W24320 /-/-/-/ SEQ ID NO: 24 (base sequence) I
  • Homo sapiens interleukin-4 induced gene-1 protein (FIGl) / 82009_at / AI85962 0 / AF293462 / MK73362 /-/ SEQ ID NO: 25 (base sequence), SEQ ID NO: 14 1 (amino acid sequence) I FLJ11436 fis, clone HEMBA1001213 / 44526_at / M191741 / AK021498 /-/-/ Sequence number: 26 (base sequence) I
  • SEQ ID NO: 31 base sequence
  • SEQ ID NO: 144 amino acid sequence
  • bone morphogenetic protein 7 osteogenesis protein 1
  • 6 osteogenesis protein 1 bone morphogenetic protein 1
  • 6steogenic protein 1 / 64137_at / AI094860 / NM_ 001719 / NP— 001710 / EMBO J. 9 (7), 2085-2093 (1990) //
  • SH3- domain protein 5 (pons in) / 45978_at / AI377221 / NM_006434 / NP_006425 / Genome Res. 11 (3), 422-435 (2001) //
  • FLJ23271 f is, clone HEP00174 / 89747— at / AI832193 /-/-/-/ SEQ ID NO: 117 (base sequence) I
  • insulin receptor substrate 2 (IRS- 2) / 56338— at / 031286 / NM— 003749 / NP— 00374 0 / J. Biol. Chem. 272 (40), 25267-25274 (1997) //
  • beta- galactose- 3-0- sulfotransf erase 4 / 44999_i_at / N37065 / M_024637 / NP_078 913 / J. Biol. Chem. 276 (28), 25697-25704 (2001) //
  • diacylglycerol 0-acyltransferase homolog 2 (mouse) / 53200_at / AA723692 / NM_0 32564 / NP— 115953 / J. Biol. Chem. 276 (42), 38870—38876 (2001) //
  • synaptotagmin 8 (L0C90019) / 55233_at / AI814253 / NM_138567 / P_612634 / Unpublis hed (2000) / SEQ ID NO: 63 (base sequence), SEQ ID NO: 149 (amino acid sequence) I hypothetical protein FLJ13881 / 59370_at / AI709055 / NM_024729 / NP_079005 / Unpu blished (2000) / SEQ ID NO: 64 (base sequence), SEQ ID NO: 150 (amino acid sequence) I
  • DNER delta-notch-like EGF repeat-containing transmembrane
  • each gene was identified as genes derived from mice that can be used as indicators of atopic dermatitis. Any of these genes can be used as an indicator gene in the present invention.
  • Each of the data for each gene ⁇ "shown below contains the following information in order from the left: Each information is separated by a slash (/). The function of each gene is It can be clarified based on the description of the gene.
  • keratin complex 2 basic, gene 6a / 104370_s_at / K02108 / NM_010669 /-/ NP_034799 /-/ Takahashi, K. et al, Genomics 53, 170—183 (1998) / AI590722 / keratin 6a / arachidonate 5-lipoxygenase activating protein Putative Ortholog / 113808 at / AA930477 /-/ 161 /-/ 167 /-/ AI983204 / arachi donate 5— lipoxygenase- activating protein (FLAP) /
  • serine protease inhibitor 2-2 Homolog / 104374_at / M64086 / NM_009252 /-/ NP_03 //: / O 6600 ⁇ 1 £ / -S0sa AV
  • RNA for gene chip analysis from human skin Preparation of RNA for gene chip analysis from human skin, cDNA synthesis for gene chip, and DNA chip analysis were performed according to Example 1.
  • GeneSpring4.2 (Silicon Genetics), a DNA chip analysis software, was used to compare gene expression in the rash-free area of each of 10 patients and gene expression in normal tissues of 6 healthy subjects. According to the GeneSpring User Manual, the result of Absolute Analysis by Affymetrix ⁇ f software Suit e4.2 is imported into GeneSpring, the average difference value of each gene for all genes on the same chip is divided by its median value, and the correction value in the chip (Correction value A) was obtained. Further, for each gene, a correction value B was obtained by dividing the correction value A for all the chips used by the median value.
  • a Mann-Whitney's U test was performed using the positive value B, and genes having a significant difference in the expression level between the non-rash area of the patient and healthy skin were selected. From the selected gene group, the average of the expression levels of each gene in the non-rash area of the patient and the healthy skin was compared, and genes having a difference of at least two times were selected. Only genes with P (present) in at least 4 out of 6 healthy subjects were selected for genes with higher expression in healthy skin compared to non-rash in patients, and genes with high expression in non-rash in patients 6 out of 10 patients Only genes that became P in the above examples were selected. The results are shown in Tables 11 to 16.
  • Table G-1 Genes whose expression was increased in the eruption of AD patients compared to normal tissues of healthy subjects
  • genes were identified as genes that can be used as indicators of atopic dermatitis. Any of these genes can be used as an indicator gene in the present invention.
  • UDP-N-acetyl-alpha-D-galactosamine polypeptide N-acetylgalactosaminyltran sf erase 7 / 75451_s— at / AW026164 I ⁇ — 054110 / NP_473451 /-
  • duodenal cytochrome b (FLJ23462) I 50955— at / AI743715 / ⁇ —024843 / NP— 0 79 119 I-
  • Example 4 Comparison of gene expression between skin tissue of atopic dermatitis patients and skin tissue of psoriatic patients.
  • the purpose of the present invention was to conduct a gene expression analysis in the skin tissue of a psoriasis patient with the aim of selecting a gene showing a specific expression change in atopic dermatitis pathology from the gene group selected in Example 1, and The results were compared with the results of gene expression analysis in the skin tissues of patients. Patients with psoriasis 6. With the consent of the patients, three skin specimens (3 mm in diameter) were collected from each part of the eruption and lesion. RNA preparation for gene chip analysis from human skin, cDNA synthesis for gene chip, and DNA chip analysis were performed according to Example 1.
  • a comparative analysis of gene expression in the rash area and rash area of 6 patients with psoriasis was performed by Compparison analysis. Genes whose expression was reduced to less than 1/2 were selected. This time, the genes selected for each patient were commonly changed in 4 or more of the 6 patients. A running gene was selected. For these gene groups, Wilcoxon signed-ranks test was performed using the Average Difference value after Comparison analysis, and the p-value was calculated.
  • Example 2 By comparing the selected gene group and the gene group whose expression change was observed between the non-rash area and the rash area of atopic dermatitis patients performed in Example 1, the common fluctuation was observed in atopic dermatitis and psoriasis Genes whose expression was fluctuated only in atopic dermatitis and genes whose expression was fluctuated only in psoriasis were identified.
  • Tables 17 to 25 list the genes that fluctuated commonly in atopic dermatitis and psoriasis, and Tables 26 to 33 show genes that showed expression changes only in atopic dermatitis. The genes showing the current variation are shown in Tables 34 to 49.
  • ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ has a variation of 50 times or more, ⁇ 10 10 to 50 times, ⁇ 3 3 to 10 times, ⁇ 2 to 3 times, and 1 has no change Is shown.

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Abstract

L'invention concerne une méthode d'examen de la dermatite atopique consistant à trouver un gène présentant une différence dans le niveau d'expression entre un site à éruption et un site exempt d'éruption chez un patient souffrant de dermatite atopique ou de psoriasis. La méthode consiste également à trouver un gène présentant une différence dans le niveau d'expression entre un site exempt d'éruption dans une telle maladie et un sujet normal. Ces gènes sont utiles comme indicateurs dans l'examen de la dermatite atopique ou du psoriasis ou dans le criblage d'un remède pour ces maladies. En résumé, la méthode d'examen de la dermatite atopique ou du psoriasis et le procédé de criblage d'un composé permettant de traiter ces maladies selon l'invention sont fondés sur la comparaison des niveaux d'expression de ces gènes indicateurs.
PCT/JP2003/009999 2002-08-06 2003-08-06 Methode d'examen de la dermatite atopique Ceased WO2004016785A1 (fr)

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US7741433B2 (en) 2005-02-24 2010-06-22 Compugen Ltd. Diagnostic markers, especially for in vivo imaging and assays and methods of use thereof
US20140187496A1 (en) * 2011-05-20 2014-07-03 Pierre Bitoun Sgef controls macular, corpus callosum and hippocampal function and development, liver homeostasis, functions of the immune system, fever response atherosclerosis and tumorogenic cell growth
CN109475108A (zh) * 2016-07-05 2019-03-15 成均馆大学校产学协力团 诱发牛皮癣动物模型及其用途
WO2020081664A1 (fr) * 2018-10-16 2020-04-23 National Jewish Health Procédés d'identification de dermatite atopique et d'allergies alimentaires
US11937937B2 (en) 2018-10-16 2024-03-26 National Jewish Health Methods of identifying atopic dermatitis and food allergies

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